Phosphatase inhibitors and methods of use thereof

ABSTRACT

The present invention is directed to compounds having the formula: ##STR1## The invention further provides a method of making the compounds. The compounds are useful as inhibitors of protein phosphatases, for example PP1, PP2A, PP3, CDC25A and CDC25B. The invention is further directed to a method of inhibiting a protein phosphatase, a method of inhibiting cell proliferation, and pharmaceutical compositions comprising the subject compounds.

This is a divisional of application Ser. No. 08/688,530 filed on Jul.30, 1996 now U.S. Pat. No. 5,700,821.

FIELD OF THE INVENTION

The regulation of protein phosphorylation by kinases and phosphatasescontrols many eukaryotic cell functions, including signal transduction,cell adhesion, gene transcription and cell proliferation. Theidentification and characterization of kinases, phosphatases, andinhibitors thereof thus allows pharmaceutical regulation of a variety ofcellular functions. The present invention provides inhibitors of proteinphosphatases, and methods of making and using the inhibitors. Thecompounds of the present invention are useful, for example, asinhibitors of cell proliferation.

BACKGROUND OF THE INVENTION

Many eukaryotic cell functions, including signal transduction, celladhesion, gene transcription, RNA splicing, apoptosis and cellproliferation, are controlled by protein phosphorylation. Proteinphosphorylation is in turn regulated by the dynamic relationship betweenkinases and phosphatases. Considerable research in synthetic chemistryhas focused on protein kinases. However, recent biological evidence formultiple regulatory functions of protein phosphatases has triggeredfurther investigation of phosphatases. The protein phosphatasesrepresent unique and attractive targets for small-molecule inhibitionand pharmacological intervention.

Most eukaryotic amino acid phosphate derivatives in polypeptides andproteins are found on serine, threonine and tyrosine residues. Threebasic types of eukaryotic protein phosphatases have been defined:serine/threonine protein phosphatases (PSTPases), tyrosine proteinphosphatases (PTPases), and dual-specificity phosphatases (DSPases). TheDSPases dephosphorylate tyrosine and threonine residues on the samepolypeptide substrate.

The serine/threonine protein phosphatases (PSTPases) are furtherclassified into subfamilies (PP1, PP2A, PP2B, PP2C and PP3) by substratespecificity, metal ion dependence and sensitivity to inhibition. Atleast forty different enzymes of this type have been identified throughDNA cloning. Potent inhibitors of the serine/threonine phosphatases havebeen identified, including proteins designated Inhibitor-1, Inhibitor-2,DARPP-32, and NIPP-1, which are reviewed for example by Honkanen et al.in Protein Kinase C, Kuo, ed., Oxford Univ. Press, Oxford, 1994, p.305.In addition, several toxins, mostly from marine organisms, have beenidentified as potent inhibitors of the serine/threonine phosphatases.The natural product inhibitors are depicted in FIG. 1 and discussed forexample by Fujiki et al. (1993) Gazz. Chim. Ital. 123: 309.

Okadaic acid, a polyether fatty acid produced by several species ofmarine dinoflagellates, reversibly inhibits the catalytic subunits ofserine/threonine phosphatase subtypes PP1, PP2A and PP3. However,okadaic acid does not rapidly penetrate cell membranes and accumulateswithin cells slowly, making it difficult to control the intracellularconcentration of the compound. Further, okadaic acid is not verychemically stable.

Other natural product inhibitors have been identified that are morestable, may penetrate some cell types better, are more potent, andexhibit selectivity toward different PSTPase isotypes. Calyculin A is acytotoxic component of the marine sponge Discodermia calyx. It has anextremely high affinity to PP1, PP2A and PP3, with an inhibitoryconcentrations₅₀ (IC₅₀, the concentration that causes 50% inhibitioncompared to untreated control preparation) being about 0.3 nM.Microcystins are potent cyclic hepta- and pentapeptide toxins of thegeneral structure cycloD-Ala-X-D-erythro-b-methyl-iso-Asp-Y-Adda-D-iso-Glu-N-methyldehydro-Ala!wherein X and Y are variable L-amino acids. Microcystins are known topromote tumors in vivo, but, with the exception of hepatocytes, areimpermeable to most cells in vitro. Compounds of the nodularin seriesexhibit IC₅₀ 's for PP1 and PP3 of about 2 and 1 nM, respectively.Motuporin, which has been recently isolated from a New Guinea sponge, iseven more potent, with an IC₅₀ of less than 1 nM for PP1. Tautomycin isproduced by a terrestrial Streptomyces strain, and inhibits PP1, PP2Aand PP3 indiscriminately with an IC₅₀ in the 15 nM range. The remainingnatural product inhibitors, thyrsiferal-23-acetate acetate andcantharidine, are somewhat selective, but weak (IC₅₀ of 0.16-10 μM),inhibitors of PP2A.

High toxicity, especially hepatotoxicity, is commonly found with thenaturally occurring serine/threonine phosphatase inhibitors. The hightoxicity appears to be intrinsically associated with non-specificphosphatase activity, and often limits the range of feasiblepharmacological studies. Honkanen (1994) Toxicon 32: 339. Further, thechemical diversity of compounds obtained from natural sources islimited. Accordingly, there is a need in the art to diversify thechemical complexity of the natural products and to optimize biochemicaland pharmacological effects.

However, only limited structure-activity relationship (SAR) studies havebeen reported on naturally occurring serine/threonine phosphataseinhibitors. For example, SAR studies of okadaic acid indicate that thecarboxyl group as well as the four hydroxyl groups are important foractivity. Nishiwki et al. (1990) Carcinogenesis 11:1837; Takai et al.(1992) Biochem J. 284:539; Sasaki et al. (1994) Biochem J. 288:259.

A limited SAR study of naturally occurring microcystins was performed byRinehart et al. (1994) J. Appl. Phycol. 6: 159. It was found that thesubstitution of alanine for arginine has little effect on phosphataseinhibitory potency, but does result in a difference in relativecytotoxicity. The dehydroamino acid residue and the N-methylsubstituents were also found to be noncritical. Esterification of theglutamic acid residue led to inactive compounds, and the (6Z) Addaisomer was inactive, suggesting the criticality of the glutamic acidunit and the overall shape of the Adda residue. However, some variationsin the Adda unit, for example the O-demethyl and the O-demethyl-O-acetylanalogs, exerted little effect on bioactivity. The general SAR of thenodularin series appears similar to the microcystins, although fewercompounds are available for testing. SAR studies have not been reportedto date for calyculin A, tautomycin or thyrsiferyl acetate.

The DSPase class of phosphatases has recently been defined, and itsmembers are emerging as important regulators of cell cycle control andsignal transduction. The first documented DSPase, VH1, as described byGuan et al. (1992) Proc. Natl. Acad. Sci. USA 89: 12175, corresponds tothe H1 open reading frame of Vaccinia virus. Other members of the DSPaseclass have been identified and generally fall into two substrate motifs,the VH1 type and the CDC25 type. Mammalian cells contain at least threecdc25 homologues (cdc25A, cdc25B and cdc25C). The CDC25 phosphatases arepositive regulators of cell cycle progression, and are reviewed byHunter et al. (1994) Cell: 573. Further, there is a strong link betweenoverexpression of the CDC25 phosphatases and oncogenic transformations,particularly in human breast cancer. Galaktinov et al.(1995) Science269: 1575. However, no potent inhibitors of the DSPases are known.

Since nearly all forms of human neoplasias have altered cell cyclecontrol, the role of phosphatases in cell cycle control makes thesemolecules attractive targets for pharmaceutical intervention. Theability of phosphatase inhibitors to interfere with aberrant cellactivity has been demonstrated. For example, the naturally occurringPSTPase inhibitor okadaic acid has been shown to induce apoptosis inmyeloid leukemia cells (Ishida et al.(1992) J. Cell. Physiol. 150: 484)and in rat hepatocytes, rat pituitary adenoma cell, human mammarycarcinoma cells and human neuroblastoma cells (Boe et al.(1991) Exp.Cell Res. 195: 237). Thus there is a significant need to design andsynthesize selective modulators of this family of enzymes in order toidentify useful therapeutic agents.

SUMMARY OF THE INVENTION

The present invention provides a compound having the formula: ##STR2##wherein R, R', R" and R'" are the same or different and are preferablyhydrophobic groups.

In another embodiment, the present invention provides a method ofsynthesizing a compound having the formula: ##STR3## wherein R, R', R"and R'" are the same or different and are preferably hydrophobic groups.

The method of synthesis comprises coupling glutamate to a solid support,adding the hydrophobic residue R'" COX, wherein X is a leaving group,adding diamine having the formula --NHCH₂ CH₂ NH(R"), adding oxazolecarboxylic acid having the formula ##STR4## wherein R and R' are thesame or different and are preferably hydrophobic groups, and cleavingthe resulting compound from the solid support.

The present invention further provides a method of inhibiting a proteinphosphatase comprising contacting a phosphatase-inhibiting effectiveamount of a compound of formula I with a protein phosphatase underconditions whereby the activity of the protein phosphatase is inhibited.

The present invention further provides a method of inhibiting cellproliferation comprising introducing into cells aproliferation-inhibiting amount of a compound of formula I. In apreferred embodiment the cells are human breast cancer cells.

Pharmaceutical compositions comprising a compound of formula I and apharmaceutically acceptable carrier are also provided by the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the formulae of naturally occurring inhibitors of theserine/threonine phosphatases.

FIG. 2 presents a representative scheme for the synthesis of compoundshaving formula I.

FIG. 3 presents a scheme for a general synthetic route to monoprotectedethylene diamines.

FIG. 4 shows a scheme for the synthesis of a heterocyclic moiety fromN-benzoyl threonine.

FIG. 5 shows a scheme for the solution phase synthesis of model compoundII.

FIG. 6 is a graph of inhibition of PP2A activity by compound 1d. Thecatalytic subunit of PP2A was incubated with vehicle alone (control),calyculin A (10 nM) or compound 1d (100 uM), and the dephosphorylationof the substrate fluorescein diphosphate determinedspectrophotometrically. Mean results of two independent experiments areshown; bars indicate the range.

FIG. 7 presents a graph depicting the ability of compounds 1a-r toinhibit CDC25A and CDC25B activity. Results are presented as percentageof control (100%).

FIG. 8 presents a dose-response curve of the ability of compound if toinhibit CDC25A.

FIG. 9 is a graph showing the anti-proliferative effect of compound ifagainst human MDA-MB-231 breast cancer cells.

FIGS. 10A-D depict cell cycle distribution of human breast cancer cellsafter treatment with compound if determined by flow cytometry. FIG. 10Ashows flow cytometry analysis of MDA-MB-231 cells treated with vehiclealone. FIG. 10B shows flow cytometry analysis 48 hours after treatmentwith 88 μM compound if. Fluorescence channel measures intracellularpropidium iodide concentration, an index of DNA content. Horizontal barsare the gating positions that allow for cell cycle analysis. FIG. 10Cshows MDA-MB-231 cell cycle distribution 48 hours after continuoustreatment with 88 μM compound if, and is the result of one experiment.Open bars are control cells and black bars are cells treated withcompound if. FIG. 10D shows cell cycle distribution 72 hours aftercontinuous treatment with 88 μM compound if. The mean values wereobtained from three independent determinations. Open bars are controlcells and black bars are cells treated with 88 μM compound if. Thestandard errors of the means are displayed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds having the formula I asfollows: ##STR5## wherein R, R', R" and R'" are the same or differentand are preferably hydrophobic groups. In a preferred embodiment, R, R',R" and R'" are independently H, alkyl, alkenyl, alkynyl, cycloalkyl,phenyl (Ph), oxetanyl, azetidinyl, furanyl, pyrrole, indolyl, oxazolyl,isoxazolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, pyranyl,pyridyl, pyridonyl, piperidyl, piperazinyl, quinolyl, azepinyl, anddiazepinyl. In another preferred embodiment, R and R'" are independentlyPh, CH₃, n-C₅ H₁₁, n-C₇ H₁₅, n-C₉ H₁₉, PhCHCH, PhCH₂ CH₂, Ph(CH₂)₂CC(CH₃), (p-MeO) Ph, (p-MeNHCO)Ph, PhCHC(CH₃)CH₂ CH₂, Ph(CH₂)₂CHCHCHC(CH₃), Ph(CH₂)₂ CHCHCHCH, Ph(CH₂)₃ CHC(CH₃)CHCH, C₆ H₁₃CH(CH₃)CHC(CH₃)CHCH, or C₄ H₉ CH(CH₃)CHC(CH₃)CHC(CH₃), R' is H, CH₃ orPh, and R" is H, CH₃, benzyl (Bn), CH₂ CH(CH₃) n-C₆ H₁₃, CH₂ CH₂ NHBn,CH₂ CH₂ Ph, or (CH₂)₃ Ph. In a most preferred embodiment, R is Ph, R' isPh, R" is Bn or CH₃ and R'" is n-C₉ H₁₉.

The compounds of the present invention may be synthesized by solid-phasecombinatorial chemistry techniques. The present invention provides amethod of synthesis of the compounds of the invention by a single-beadcombinatorial strategy whereby the structure of each compound is known.The present method thus avoids the difficulties inherent in other priorart combinatorial syntheses, for example the need for sophisticatedtagging schemes or extensive analytical techniques to identify thesynthesized compounds.

The method of synthesizing the compounds of the present inventioncomprises coupling a diprotected glutamate moiety to a solid support,deprotecting the glutamate amino-terminus, adding the hydrophobicresidue R'" COX, wherein X is a leaving group such as chloride,anhydride, active ester, pentafluorophenyl, phosphate derivative orphosphonate derivative, deprotecting the glutamate carboxy-terminus,adding protected diamine having the formula A-NHCH₂ CH₂ NH(R") wherein Ais a protection group, deprotecting the amino-terminus of the diamine,adding an oxazole carboxylic acid having the formula ##STR6## andcleaving the resulting compound from the solid support. By couplingprotected glutamate to the solid support on a large scale, anddistributing batches of the growing compound to different vessels ateach step, for example after the removal of each protecting group, alarge number of compounds can be synthesized during each reaction run byvarying the R, R', R" and R'" groups. A representative synthetic schemeis set forth in FIG. 2.

Solid supports for combinatorial synthesis are known to those ofordinary skill in the art. In a preferred embodiment of the presentinvention, the solid support is a polystyrene resin. In a more preferredembodiment, the solid support is the polystyrene resin described by Wang(1973) J. Amer. Chem. Soc. 95: 1328, and also known as the Wang resin.The Wang resin is commercially available, for example from AdvancedChemtech. Other suitable solid supports include polyethyleneglycol-polystyrene graft or Rink resins.

Diprotected glutamate may be prepared by methods known in the art. Theskilled artisan is aware of suitable protecting groups for the amino-and carboxy-termini of glutamate, which are reviewed for example byGreene et al. Protective groups in organic synthesis, 2nd edition,Wiley, N.Y., 1991. In a preferred embodiment the amino-terminalprotecting group is Fmoc and the carboxyl-terminal protecting group is aγ-allyl ester group. This preferred diprotected glutamate may besynthesized by protecting the carboxylic function with the allyl esteras described by Belshaw et al. (1990) Syn Comm. 20: 3157, followed bytreatment with Fmoc-Cl. According to the method of Belshaw et al.,chlorotrimethylsilane is added to a suspension of L-glutamic acid in dryallyl alcohol under N₂ and stirred for 18 hours, followed by theaddition of ethyl ether.

Diprotected glutamate is coupled to a solid support by methods known tothose of ordinary skill in the art and appropriate for the desiredsupport. In a preferred embodiment, diprotected glutamate is coupled tothe Wang resin with 1-ethyl-3- 3-(dimethylamino)propyl!-carbodiimidehydrochloride (EDCl) on a large scale to provide a supply of solid phasebeads coupled to diprotected glutamate. Other suitable coupling methodsare reviewed for example by Bodanszky, Principles of peptide synthesis,2nd edition, Springer, Berlin, 1993, and include DCC, HOBt, BOCreagents, and others.

The glutamate amino-terminus is deprotected by a method appropriate tothe protecting group. For example, a base labile group such as Fmoc maybe removed by treatment with piperidine and tetrahydrofuran (THF). Atthis point the resin may be distributed to a number of separate vesselsin order that compounds having different R'" substituents may beprepared. For example, the resin may be distributed to multiple filtersequipped with inert gas inlets for maintaining steady bubbling andsuction adapters. After the addition of solvent, hydrophobic residuesR'" COX are added to each flask. Thus different amide derivatives areprovided, the number of which is determined by the number of vessels towhich the resin has been distributed and the number of different R'"substituents. By adding R'" COX having differing R'" groups to eachvessel, the final compounds can be conveniently identified. R'" CO₂ H orR'" COX may be prepared by standard solution synthesis or obtainedcommercially.

After filtration and rinsing of the solid support, the carboxy-terminalprotecting group is removed by a method suitable for the protectinggroup. For example, allyl esters may be removed by Pd(O) chemistry asdescribed by Dangles et al. (1987) J. Org. Chem. 52: 4984. The resin mayagain be divided into batches at this point and distributed intoseparate vessels such as the filters described above so that compoundscontaining different R" groups may be prepared. The protected diamine isthen added with a suitable coupling agent so that the side chaincarboxyl terminus of glutamate is extended. In a preferred embodimentthe protecting group of the diamine is an N-allyloxycarbonyl group suchthat the diamine has the formula Alloc-NHCH₂ CH₂ NH(R"). Suitablecoupling agents include for example PyBroP⁶⁰ and CloP⁶¹. Alloc-NHCH₂ CH₂NH(R") may be conveniently prepared by carbamoylation ofchloroethylamine followed by treatment with sodium iodide andcommercially available amine R"NH₂, as depicted in FIG. 3.

The resulting compounds are deprotected, at which point the compoundsmay be again distributed to different vessels for the addition ofdifferent R and R' substituents. Coupling with oxazole carboxylic acidshaving the formula ##STR7## wherein R and R' are as defined above forformula I, in the presence of CloP, followed by rinsing with solventprovides the compounds of the invention attached to the solid support.The oxazole carboxylic acids may be prepared separately in solutionphase from carboxylic acids having the structure R--CO₂ H wherein R isas defined above for formula I and serine methyl ester, threonine methylester and phenyl serine methyl ester. An oxidation-cyclodehydrationprotocol depicted in FIG. 4 and described by Wipf et al. (1993) J. Org.Chem. 58: 3604, followed by saponification yields the desired carboxylicacid segments. The intermediate oxazole esters may be purified by columnchromatography on SiO₂.

The carboxylate may be released from the support by complete or partialcleavage with 50% trifluoroacetic acid to provide the compounds of theinvention. After filtration of the solid support and evaporation of theresulting mother liquor, the compounds of Formula I are chemically pureand structurally well-defined.

It has been found in accordance with the present invention thatcompounds of formula I are capable of inhibiting serine/threonineprotein phosphatases. Inhibition of serine/threonine proteinphosphatases is defined herein as inhibition of the activity of one ormore of PP1, PP2A or PP3 at a concentration of 100 μM or less of theinhibitor compound. In a preferred embodiment, the activity of thephosphatase is inhibited by at least 10%. In more preferred embodiments,the activity of the phophatase is inhibited by at least 25%, or evenmore preferably, by at least 50%. Inhibition of PP1, PP2A or PP3 can beassessed by methods known to one of ordinary skill in the art. Suitableassays are described, for example by Honkanen et al.(1994) Toxicon32:339 and Honkanen et al. (1990) J. Biol. Chem. 265: 19401, thedisclosures of which are incorporated herein by reference. Briefly,phosphatase activity is determined by quantifying the ³² P! releasedfrom a ³² P-labeled substrate such as phosphohistone or phosphorylase-a.Decreased ³² P! release in the presence of the compounds of the presentinvention relative to control samples provides a measure of the abilityof the compounds of the invention to inhibit PP1, PP2A or PP3.

In another suitable assay, the ability of the compounds of the presentinvention to inhibit the activity of protein phosphatase PP2A isassessed. The activity of the catalytic subunit of bovine cardiac musclePP2A (Gibco-BRL, Gaithersburg, Md.) is measured in 96-well microtiterplates using the substrate fluorescein diphosphate as follows.Inhibitors are resuspended in DMSO, which is also used as the vehiclecontrol. An incubation mixture of 150 μL is prepared containing 25 mMTris, pH 8.0, 5 mM EDTA, 33 μg/ML BSA, 20 μM fluorescein diphosphate,and 100 μM inhibitor or DMSO control. Reactions are initiated by adding0.2 units of PP2A , and incubated at room temperature overnight.Fluorescence emission from the product is measuredspectrofluorometrically, for example with Perseptive BiosystemsCytofluor II (Framingham, Mass.) (excitation filter, 485 nm; emissionfilter, 530 nm). The rate of increase in absorbance due to formation ofdephosphorylated substrate is proportional to phosphatase activity.Thus, decreased absorbance relative to control samples provides ameasure of the ability of the present compounds to inhibit thephosphatase.

The compounds of the present invention are also capable of inhibitingdual specificity phosphatases, for example CDC25A and CDC25B.Phosphatases CDC25A and CDC25B are disclosed in U.S. Pat. No. 5,441,880.Inhibition of dual specificity phosphatases is defined herein asinhibition of the activity of CDC25A and/or CDC25B at a concentration of100 μM of the inhibitor compound. In a preferred embodiment, theactivity of the phosphatase is inhibited by at least 10%. In morepreferred embodiments, the activity of the phophatase is inhibited by atleast 25%, or even more preferably, by at least 50%. The ability of thecompounds of the present invention to inhibit the phosphatases can bedetermined by measuring the effect of the compounds on the ability ofCDC25 or CDC25B to dephosphorylate a substrate. Appropriate methods areknown to those of ordinary skill in the art, and include, for example,calorimetric assays. A suitable assay is described in U.S. Pat. No.5,441,880, the disclosure of which is incorporated herein by reference.As disclosed therein, the compound to be tested is combined with CDC25Aor CDC25B and an appropriate CDC25 substrate, such as p-nitrophenylphosphate or inactive cyclin/cdc2, and the ability of the compound toinhibit the phosphatase activity of CDC25 is assessed. Phosphataseactivity may be assessed by known techniques, such as measuring opticaldensity and comparing it to the optical density of a control sample thatdoes not contain the inhibitor. The assay may be performed as a rapidcalorimetric microtitration plate assay.

The compounds of the present invention are useful as inhibitors ofprotein phosphatases. It is known that inhibition of proteinphosphatases results in increased protein phosphorylation in vitro andin cells. Sassa et al. (1989) Biochem. Biophys. Res. Comm. 159: 939;Yatsunami et al.(1991) Biochem. Biophys. Res. Commun. 177: 1165. Thusthe compounds are useful to inhibit protein dephosphorylation, forexample in in vitro assays in which phosphorylated proteins are measuredor detected, or in methods in which proteins are labeled byphosphorylation. For example, proteins are commonly labeled with ³² P tofacilitate detection. Inclusion of a compound of the present inventionis useful to prevent dephosphorylation by endogenous phosphatases.Further, phosphatases are known to have multiple functions, includingbut not limited to regulation of signal transduction, cell adhesion,gene transcription, RNA splicing, apoptosis, mitosis and cellproliferation. Thus inhibitors of protein phosphatases are useful in thealteration of various cellular functions. In particular, the presentcompounds are useful as inducers of apoptosis and inhibitors of cellproliferation.

Accordingly, the present invention provides a method of inhibiting aprotein phosphatase comprising contacting a phosphatase-inhibitingeffective amount of a compound having the formula: ##STR8## with aprotein phosphatase under conditions whereby the activity of the proteinphosphatase is inhibited. In a preferred embodiment, R, R', R" and R'"are independently H, alkyl, alkenyl, alkynyl, cycloalkyl, phenyl (Ph),oxetanyl, azetidinyl, furanyl, pyrrole, indolyl, oxazolyl, isoxazolyl,imidazolyl, pyrazolyl, triazolyl, tetrazolyl, pyranyl, pyridyl,pyridonyl, piperidyl, piperazinyl, quinolyl, azepinyl, and diazepinyl.In another preferred embodiment, R and R'" are independently Ph, CH₃,n-C₅ H₁₁, n-C₇ H₁₅, n-C₉ H₁₉, PhCHCH, PhCH₂ CH₂, Ph(CH₂)₂ CC(CH₃),(p-MeO)Ph, (p-MeNHCO)Ph, PhCHC(CH₃)CH₂ CH₂, Ph(CH₂)₂ CHCHCHC(CH₃),Ph(CH₂)₂ CHCHCHCH, Ph(CH₂)₃ CHC(CH₃)CHCH, C₆ H₁₃ CH(CH₃)CHC(CH₃)CHCH, orC₄ H₉ CH(CH₃)CHC(CH₃)CHC(CH₃), R' is H, CH₃ or Ph, and R" is H, CH₃,benzyl (Bn), CH₂ CH(CH₃), n-C₆ H₁₃, CH₂ CH₂ NBn, CH₂ CH₂ Ph, or (CH₂)₃Ph. In a most preferred embodiment, R is Ph, R' is Ph, R" is Bn or CH₃and R'" is n-C₉ H₁₉. In a preferred embodiment the protein phosphataseis a serine/threonine phosphatase. In a more preferred embodiment theprotein phosphatase is PP1, PP2A or PP3. In another preferred embodimentthe phosphatase is a dual specificity phosphatase, including, forexample, CDC25A and CDC25B. The skilled artisan readily can determinethe amount of the phosphatase inhibitor that is required to inhibitprotein phosphatase by measuring phosphatase activity in the presenceand absence of the inhibitor. Phosphatase activity can be determined byassessing the dephosphorylation of a substrate as described hereinabove,or by measuring parameters that are known to result from phosphataseactivity. For example, phosphatase inhibition by the compounds of thepresent invention can be assessed by measuring inhibition of cellproliferation in response to treatment of cells with the presentcompounds.

The compounds of the present invention are also useful asantiproliferative agents. The CDC25 enzymes are positive regulators ofcell cycle progression. For example, CDC25C drives entry into mitosis bydephosphorylating and thereby activating the mitotic inducer cdc2. U.S.Pat. No. 5,294,538 discloses that dephosphorylation of cdc2 by the CDC25phosphatase activates the M phase-promoting factor (MPF) that triggersthe G2/M transition of the cell cycle, and that inhibition of the CDC25phosphatase activity inhibits entry of cells into mitosis. Further,CDC25A and CDC25B act as oncogenes, and CDC25B is over-expressed in onethird of primary human breast cancers (Galaktionov et al., (1995)Science 269: 1575) and is elevated in other human tumor cell types andin virally transformed cells (Nagata et al.(1991) New Biologist 3:959).It is also known that cell proliferation is coordinated bycyclin-dependent kinases, and tightly controlled by both kinases andphosphatases. The cell cycle regulating activity of CDC25 is controlledby PP2A. Hunter et al.(1994) Cell 79: 573. Thus, inhibition of PP1 orPP2A may also result in disrupted cell cycle transition. It has beendemonstrated in accordance with the present invention that compounds ofthe invention inhibit proliferation of cells by reducing the number ofcells in inhibitor-treated versus untreated cell cultures. Accordingly,the present invention further provides a method of inhibiting cellproliferation comprising introducing into cells aproliferation-inhibiting amount of a compound of formula I. In apreferred embodiment the compound has formula I wherein R is Ph, R' isPh, R" is Bn and R'" is n-C₉ H₁₉. In another preferred embodiment thecells are tumor cells. In still another preferred embodiment the cellsare human breast cancer cells.

The ability of the compounds of the present invention to inhibitproliferation can be assessed by methods known to those of ordinaryskill in the art. For example, proliferating cells can be contacted witha compound of the invention, and cell numbers determined. A reduction incell number in treated versus untreated cells provides a measure of theability of the present compounds to inhibit proliferation. In apreferred embodiment the cells to be treated are cancer cells, forexample breast cancer cells such as the CDC25B+ breast cancer cellsMDA-MB-231, which are available from the American Type CultureCollection (Accession No. HTB-26). The antiproliferative activity of thepresent compounds can also be measured by the assay described by Lazo etal.(1995) J. Biol. Chem. 270: 5506, the disclosure of which isincorporated herein by reference. The microtiter-based calorimetricassay is based upon the reduction of 3-4,5-dimethylthiazol-2-yl!-2,5-diphenyl tetrazolium bromide by livingcells, and permits evaluation of the cytostatic or cytotoxic actions oflarge numbers of compounds quickly in mouse embryonic fibroblasts andhuman CDC25B+ breast cancer cells. Briefly, exponentially growingCDC25B+ MDA-MB-231 cells are treated continuously with from 0 to 100 μMof a compound of the invention, and cell proliferation is determinedcalorimetrically after 72 hours by assessing the ability of the cells toreduce 3- 4,5-dimethylthiazol-2-yl!-2,5-diphenyl tetrazolium bromide.

The antiproliferative ability of the compounds of the present inventioncan also be measured in an in vivo tumor reduction assay. Athymic(nu/nu) and severe combined immunodeficiency (SCID) mice providewell-recognized models for antitumor activity. Mice are injectedsubcutaneously (s.c.) with 10⁶ MDA-MB-231 cells in 100 μl phosphatebuffered saline (PBS). Once tumors reach a palpable size (100 mm²), miceare treated once daily orally (p.o.), intraperitoneally (i.p.) orsubcutaneously (s.c.) with 0, 0.1, 1, 10 or 30 mg/kg of a compound ofthe present invention for five days. Tumor mass is measured with avernier caliper and calculated as described by Jani et al. (1992) CancerRes. 52: 2931. Reduction in tumor mass in treated versus untreated miceis indicative of antiproliferative activity of the compounds of theinvention. Pharmacological principles may be used to optimize the doseand schedule of administration and are readily within the skill of thosein the art.

The present invention also provides pharmaceutical compositionscomprising a compound of the present invention and a pharmaceuticallyacceptable carrier or diluent. As used herein, the term"pharmaceutically acceptable carrier or diluent" means any and allsolvents, dispersion media, antibacterial and antifungal agents,microcapsules, liposomes, cationic lipid carriers, isotonic andabsorption delaying agents and the like which are not incompatible withthe active ingredients. The use of such media and agents forpharmaceutically active substances is well known in the art.Supplementary active ingredients may also be incorporated into thecompositions and used in the methods of the invention.

The formulation of pharmaceutical compositions is generally known in theart and reference can conveniently be made to Remington's PharmaceuticalSciences, 17th ed., Mack Publishing Co., Easton, Pa. Formulation of thecompounds of the present invention must be stable under the conditionsof manufacture and storage and also must be preserved against thecontaminating action of microorganisms such as bacteria and fungi.Prevention against microorganism contamination can be achieved throughthe addition of various antibacterial and antifungal agents.

The pharmaceutical forms of the compounds of the invention suitable foradministration include sterile aqueous solutions or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersions. Typical carriers include a solvent ordispersion medium containing, for example, water buffered aqueoussolutions (i.e., biocompatible buffers), ethanol, polyols such asglycerol, propylene glycol, polyethylene glycol, suitable mixturesthereof, surfactants, and vegetable oils. Isotonic agents such as sugarsor sodium chloride may be incorporated into the subject compositions.

The compounds of the invention are compounded for convenient andeffective administration in effective amounts with a suitablepharmaceutically acceptable carrier, preferably in dosage unit form.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for a subject to be treated, each unitcontaining a predetermined quantity of a compound of the inventioncalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier. The compositions may beadministered in a manner compatible with the dosage formulation, in suchamount as will be therapeutically effective, and in any way which ismedically acceptable. Possible administration routes include oral routeand parenteral administration such as intravascular, intravenous,intraarterial, subcutaneous, intramuscular, intratumor, intraperitoneal,intraventricular and intraepidural. The compositions may also bedirectly applied to tissue surfaces. Sustained release administration,for example by depot injections or erodible implants, is alsospecifically included.

The invention is further illustrated by the following specific exampleswhich are not intended in any way to limit the scope of the invention.

EXAMPLE I

A method for the combinatorial synthesis of compounds of formula I (FIG.2) was developed by optimizing a solution phase synthesis of modelcompound II. The solution phase synthesis of model compound II isdepicted in FIG. 5 and proceeded as follows:

All glassware was dried in an oven at 150° C. prior to use. THF anddioxane were dried by distillation over Na/benzophenone under a nitrogenatmosphere. Dry CH₂ Cl₂, DMF and CH₃ CN were obtained by distillationfrom CaH₂.

L-Glutamic acid (3) was protected in 62% yield as the γ-allyl esterusing allyl alcohol and chlorotrimethylsilane to provide2-amino-pentanedioic acid 5-allyl ester 4 as follows:

To a stirred suspension of 2.5 g (16.9 mmol) of L-glutamic acid (3) in40 mL of dry allyl alcohol was added dropwise 5.4 mL (42.3 mmol) ofchlorotrimethylsilane. The suspension was stirred at 22° C. for 18 h andpoured into 300 mL of Et₂ O. The resulting white solid was filtered off,washed with Et₂ O, and dried in vacuo to provide 3.80 g (62%) of ester4: Mp 133-134.5° C. (Et₂ O); IR (KBr) 3152, 2972, 2557, 1738, 1607,1489, 1450, 1289, 1366, 1264, 1223, 1177, 1146, 121, 1084 cm⁻¹ ; ¹ H NMR(D₂ O) δ 5.8-5.7 (m, 1 H), 5.14 (dd, 1 H, J=1.4, 17.3 Hz), 5.09 (dd, 1H, J=1.0, 10.4 Hz), 4.44 (d, 2 H, J=5.6 Hz), 3.92 (t, 1 H, J=6.8 Hz),2.48 (t, 2 H, J=7.0 Hz), 2.1-2.0 (m, 2 H); ¹³ C NMR (DMSO-d₆) δ 171.5,170.6, 132.7, 117.9, 64.7, 51.2, 29.3, 25.2; MS (El) m/z (relativeintensity) 188 (63), 142 (72), 128 (27), 100 (21), 85 (100), 74 (32), 56(73).

Treatment with Fmoc as follows provided2-(9-H-Fluoren-9-ylmethoxycarbonylamino)-pentanedioic acid 5-allyl ester5. To 20 mL of dioxane was added 1.5 g (6.7 mmol) of ester 4. Theresulting suspension was treated with 16.8 mmol (17.7 mL of a 10%solution) of sodium carbonate at 0° C., stirred for 5 min and treatedwith 1.74 g (6.7 mmol) of Fmoc-Cl dissolved in 10 mL of dioxane. Thereaction mixture was warmed to 22° C., stirred for 3 h, poured into 50mL of H₂ O and extracted with Et₂ O (2×25 mL). The aqueous layer wascooled to 0° C., acidified to pH 1 with concentrated HCl, and extractedwith EtOAc (3×25 mL). The resulting organic layer was dried (Na₂ SO₄)and concentrated in vacuo to give 2.72 g (99%) of 5 as a viscous oil:α!_(D) +8.5° (c 2.8, CHCl₃, 21° C.); IR (neat) 3312, 3061, 2951, 2361,2349, 2332, 1725, 1528, 1447, 1414, 1325, 1254, 1117, 1078, 1049 cm⁻¹ ;¹ H NMR δ 11.09 (bs, 1 H), 7.73 (d, 2 H, J=7.5 Hz), 7.57 (d, 2 H, J=5.1Hz), 7.4-7.25 (m, 4 H), 6.0-5.85 (m, 1 H), 5.76 (d, 1 H, J=8.1 Hz), 5.30(d, 1 H, J=19.5 Hz), 5.21 (d, 1 H, J=10.5 Hz), 4.6-4.35 (m, 5 H), 4.19(t, 1 H, J=6.6 Hz), 2.5-2.2 (m, 4 H); ¹³ C NMR δ 175.6, 172.6, 156.2,143.7, 143.5, 141.2, 131.7, 127.6, 127.0, 125.0, 119.9, 118.4, 67.1,65.4, 53.1, 46.9, 30.2, 27.1; MS (El) m/e (relative intensity) 409 (7),351 (19), 338 (12), 280 (11), 239 (11), 196 (12), 178 (100), 165 (40);HRMS (El) calculated for C₂₃ H₂₃ NO₆ : 409.1525, found: 409.1501.

Treatment with Fmoc-Cl followed by coupling to benzyl alcohol using1-ethyl-3- 3-(dimethylamino)propyl!-carbodiimide by! hydrochloride(EDCL) provided 2-(9H-Fluoren-9-ylmethoxycarbonylamino)-pentanedioicacid 5-allyl ester 1-benzyl ester 6 in 82% yield. To a solution of 1.5 g(36.6 mmol) of 5 in 5 mL of CH₂ Cl₂ was added 0.42 mL (40.3 mmol) ofbenzyl alcohol, 0.912 g (47.6 mmol) of EDCI, and 45 mg (3.66 mmol) ofdimethylaminopyridine (DMAP). The reaction mixture was stirred at 22° C.for 6 h, diluted with 20 mL of CH₂ Cl₂, and extracted with H₂ O (1×15mL), 0.1M HCl (2×15 mL), and brine (2×10 mL). The organic layer wasdried (Na₂ SO₄), concentrated in vacuo, and chromatographed on SiO₂(Hexanes/EtOAc, 5:1) to give 1.83 g (82%) of 6 as a white solid: Mp66.2-67.1° C. (EtOAc/Hexanes); α!_(D) +1.40° (c 1.64, CHCl₃, 21° C.); IR(neat) 3314, 1726, 1682, 1527, 1443, 1414, 1383, 1254, 1173, 1099, 1082,980, 754, 735 cm⁻¹ ; ¹ H NMR δ 7.75 (d, 2 H, J=7.4 Hz), 7.59 (d, 2 H,J=7.1 Hz), 7.41-7.27 (m, 9 H), 5.95-5.85 (m, 1 H), 5.44 (d, 1 H, J=8.2Hz), 5.34-5.19 (m, 4 H), 4.56 (d, 2 H, J=5.6 Hz), 4.5-4.4 (m, 3 H), 4.21(t, 1 H, J=7.0 Hz), 2.5-2.0 (m, 4 H); ¹³ C NMR δ 172.2, 171.6, 155.8,143.7, 143.5, 141.1, 135.0, 131.8, 128.5, 128.3, 128.1, 127.6, 126.9,124.9, 119.8, 118.3, 67.2, 66.9, 66.2, 53.3, 47.0, 28.0, 27.3; MS (FAB,MNBA/MeOH) m/z (relative intensity) 500 ( M+H!⁺, 40), 465 (8), 448 (14),433 (12), 413 (8), 386 (38), 371 (24), 349 (9), 324 (16), 309 (26), 293(11), 265 (10), 247 (24), 231 (56), 215 (39), 202 (26), 191 (24), 179(67), 165 (48), 154 (67), 143 (31), 133 (71), 117 (100).

The Fmoc protective group was subsequently removed by exposure to DMAPand the free amine was acylated in situ with decanoyl chloride to give2-Decanoylamino-pentanedioic acid 5-allyl ester 1-benzyl ester (7) in63% yield as follows. To a suspension of 1 g (2.0 mmol) of 6 in 10 mL ofCH₂ Cl₂ was added 1 g (8.2 mmol) of DMAP. The reaction mixture wasstirred at 22° C. for 24 h, treated with 0.62 mL (3.0 mmol) of decanoylchloride, stirred for 2 h at 22° C., and extracted with saturated sodiumbicarbonate solution (2×10 mL). The organic layer was dried (Na₂ SO₄),evaporated to dryness, and the residue was chromatographed on SiO₂(Hexanes/EtOAc, 5:1) to give 548 mg (63%) of 7 as a viscous oil: IR(neat) 3293, 3063, 2924, 2855, 1740, 1649, 1534, 1453, 1379, 1175, 986,930 cm⁻¹ ; ¹ H NMR δ 7.26 (s, 5 H), 6.68 (d, I H, J=7.8 Hz), 5.85-5.75(m, 1 H), 5.22 (d, 1 H, J=17.3 Hz), 5.14 (d, 1 H, J=10.4 Hz), 5.08 (s, 2H), 4.63-4.57 (m, 1 H), 4.48 (d, 2 H, J=5.6 Hz), 2.38-2.28 (m, 2 H),2.2-2.1 (m, 3 H), 2.0-1.9 (m, 1 H), 1.55 (t, 2 H, J=6.9 Hz), 1.20 (bs,12 H), 0.82 (t, 3 H, J=5.9 Hz); ¹³ C NMR δ 173.0, 172.1, 171.6, 135.0,131.7, 128.2, 128.1, 127.8, 117.9, 66.8, 64.9, 51.3, 36.0, 31.6, 29.9,29.1, 29.0, 26.8, 25.3, 22.3, 13.8; MS (El) m/z (relative intensity) 431(12), 319 (21), 296 (51), 142 (100), 124 (31), 91 (91); HRMS (El) m/zcalculated for C₂₅ H₃₇ NO₅ : 431.2672, found: 431.2673.

Pd(O)-catalyzed deprotection of the allyl ester proceeded as follows toyield 2-Decanoylamino-pentanedioic acid 1-benzyl ester (8). To asolution of 752 mg (1.74 mmol) of 2-decanoylamino-pentanedioic acid 7 in10 mL of CH₂ Cl₂ was added 100 mg (0.087 mmol) oftetrakistriphenylphosphine Pd(O) followed by 0.52 mL (1.9 mmol) oftributyltin hydride. After 15 min, the reaction mixture was quenchedwith 10 mL of a 10% HCl solution. The aqueous layer was reextracted with15 mL of CH₂ Cl₂ and the organic layer dried (Na₂ SO₄), concentrated invacuo, and chromatographed on SiO₂ (Hexanes/EtOAc, 9:1) to provide 545mg (79.9%) of 8 as a thick oil: α!_(D) +2.8° (c 1.2, CHCl₃, 21° C.); IR(neat) 3351, 3064, 2995, 2852, 1738, 1712, 1657, 1536, 1454, 1380, 1364,1265, 1209, 1183, 1121, 739 cm⁻¹ ; ¹ H NMR δ 10.9-10.7 (bs, 1 H), 7.22(s, 5 H), 6.58 (d, 1 H, J=7.8 Hz), 5.09 (s, 2 H), 4.63 (dd, 1 H, J=8.1,12.9 Hz), 2.4-2.25 (m, 2 H), 2.2-2.1 (m, 3 H), 2.0-1.9 (m, 1 H), (m, 6H), 1.53 (t, 2 H, J=6.6 Hz), 1.19 (bs, 12 H), 0.81 (t. 3 H, J=6.0 Hz);¹³ C NMR δ 176.9, 174.0, 171.8, 134.9, 128.5, 128.4, 128.1, 67.3, 51.4,36.2, 31.7, 29.9, 29.3, 29.2, 29.1, 27.0, 25.5, 22.5, 14.0; MS (El) m/z(relative intensity) 391 (54), 373 (62), 279 (13), 256 (19), 178 (27),178 (23), 155 (13), 146 (6), 130 (7), 102 (100); HRMS (El) m/zcalculated for C₂₂ H₃₃ NO₅ :391.2358, found: 391.2350.

Coupling to ethylene diamine (9) yielded 4-(2-Allyloxycarbonylamino-ethyl)-methyl-carbamoyl!-2-decanoylamino-butyricacid benzyl ester (10). To a solution of 526 mg (1.3 mmol) of 8 in 10 mLof CH₂ Cl₂ was added 225 μL (1.61 mmol) of triethylamine and 320 mg (2.0mmol) of secondary amine 9. The solution was stirred at 22° C. for 5min, treated with 710 mg (1.61 mmol) of benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP reagent), stirred at22° C. for 10 min, concentrated in vacuo, dissolved in 15 mL of EtOAc,and extracted with 2M HCl solution. The organic layer waschromatographed on SiO₂ (Hexanes/EtOAC, 1:3) to give 715 mg (94%) of 10as a clear oil: α!_(D) +5.3 (c 0.58, CHCl₃, 21° C.); IR (neat) 3420,3250, 2924, 1713, 1680, 1657, 1642, 1632, 1537, 1495, 1470, 1455, 1252,845 cm⁻¹ ; ¹ H NMR δ 7.35-7.2 (bs, 5 H), 6.97 (d, 0.3 H, J=7.5 Hz), 6.82(d, 0.7 H, J=7.3 Hz), 5.9-5.6 (m, 2 H), 5.3-5.1 (m, 4 H), 4.65-4.5 (m, 1H), 4.50 (d, 2 H, J=4.9 Hz); 3.55 (t, 1 H, J=7.0 Hz), 3.35-3.1 (m, 3 H),2.85 (s, 3 H), 2.4-1.8 (m, 6 H), 1.65-1.5 (m, 2 H), 1.22 (bs, 12 H),0.84 (t, 3 H, J=6.1 Hz); ¹³ C NMR (MEOD) δ 176.4, 176.3, 174.4, 174.2,173.2, 158.6, 137.1, 134.3, 134.2, 132.9, 129.5, 129.2, 129.1, 117.6,117.4, 67.8, 66.3, 66.2, 53.5, 53.3, 39.6, 39.3, 36.7, 36.6, 34.2, 32.9,30.5, 30.4, 30.3, 30.2, 29.7, 27.6, 26.8, 23.6, 14.5; MS (El) m/z(relative intensity) 531 (16), 473 (37), 418 (16), 396 (26), 374 (38),361 (17), 338 (87), 220 (54), 184 (52), 155 (36), 130 (29), 101 (37), 91(100); HRMS (El) m/z calculated for C₂₉ H₄₅ N₃ O₆ :531.3308, found:531.3316.

Monoprotected ethylene diamines 9 were easily achieved by carbamoylationof 2-chloroethylamine monohydrochloride (12), Finkelstein reaction, andaminolysis (FIG. 3).

(2-Chloro-ethyl)-carbamic acid allyl ester (13) was synthesized asfollows. A solution of 2.5 g (22 mmol) of chloroethylamine hydrochloridein 10 mL of 6M NaOH was cooled to 0° C. and treated dropwise with 2.7 mL(25.9 mmol) of allyl chloroformate while keeping the pH at 9 by additionof 6M NaOH solution. The reaction was then warmed to 22° C., stirred for2 h, and extracted with THF. The organic layer was dried (Na₂ SO₄,concentrated in vacuo, and chromatographed on SiO₂ (Hexanes/EtOAc, 9:1)to give 3.1 g (88%) of 13 as a yellow oil: IR (neat) 3333, 2949, 2348,1705, 1647, 1529, 1433, 1368, 1248, 1190, 1144, 1061, 991, 929, 776 cm⁻¹; ¹ H NMR δ 6.05-5.85 (m, 1 H), 5.55-5.35 (bs, 1 H), 5.26 (dd, 1 H,J=1.5, 17.1 Hz), 5.18 (dd, 1 H, J=1.0, 10.4), 4.54 (d, 2 H, J=5.5 Hz),3.57 (t, 2 H, J=5.5 Hz), 3.5-3.35 (m, 2 H); ¹³ C NMR δ 156.0, 132.5,117.7, 65.6, 43.8, 42.7.

To produce (2-Methylamino-ethyl)-carbamic acid allyl ester (9), asolution of 14 g (86 mmol) of 13 and 25 g (172 mmol) of sodium iodide in40 mL of acetone was refluxed for 18 h, concentrated in vacuo, dissolvedin H₂ O, and extracted with CH₂ Cl₂. The organic layer was dried (Na₂SO₄) and cooled to 0° C. Methyl amine was bubbled through the reactionmixture until the solution was saturated. The reaction mixture waswarmed to 22° C., stirred for 36 h, concentrated in vacuo andchromatographed on SiO₂ (EtOAc) to produce 6.14 g (45%) of 9 as a yellowoil: IR (neat) 3306, 2938, 2313, 1844, 1703, 1651, 1525, 1460, 1383,1256, 1144, 995, 927, 775 cm⁻¹ ; ¹ H NMR δ 5.95-5.8 (m, 1 H), 5.28 (dd,1 H, J=1.4, 17.3 Hz), 5.18 (d, 1 H, J=10.4 Hz), 4.54 (d, 2 H, J=5.3 Hz),4.9-4.6 (bs, 1 H), 3.34 (q, 2 H, J=5.6 Hz), 2.79 (t, 2 H, J=5.6 Hz),2.47 (s, 3 H); ¹³ C NMR δ 157.22, 132.8, 117.6, 65.5, 50.7, 39.7, 35.4;MS (El) m/e (relative intensity) 158 (32), 138 (17), 129 (25), 101 (13),84 (12), 73 (13), 57 (100).

The heterocyclic moiety 11 was prepared from N-benzoyl threonine (14) asfollows and as depicted in FIG. 4.

5-Methyl-2-phenyl-oxazole-4-carboxylic acid methyl ester (15) wassynthesized by treating a solution of 750 mg (3.2 mmol) of 14 in 10 mLof CH₂ Cl₂ with 1.61 g (3.8 mmol) of Dess-Martin reagent. The reactionwas stirred at 22° C. for 10 min, concentrated in vacuo, andchromatographed on SiO₂ (Hexanes/EtOAc, 3:2) to give 658 mg (89%) of2-benzoylamino-3-oxo-butyric acid methyl ester. Alternatively, asolution of 9.12 g (38 mmol) of 14 in 80 mL of CH₂ Cl₂ was cooled to-23° C. and treated with 16.1 mL (115-mmol) of triethylamine and asolution of 18.3 g (115 mmol) of SO₃ -pyridine complex in 60 mL of dryDMSO. The reaction mixture was warmed to 22° C., stirred for 30 min,then cooled to -48° C. and quenched with 20 mL of saturated NaHCO₃. Thesolution was extracted with 50 mL of Hexanes/EtOAc, 2:1. The aqueouslayer was reextracted with Hexanes/Et₂ O, 2:1, and the combined organiclayers were washed with brine, dried (Na₂ SO₄), and chromatographed(Hexanes/EtOAc, 3:2) to give 7.1 g (79%) of 2-benzoylamino-3-oxo-butyricacid methyl ester as a white solid: Mp 112.7-113.3° C. (Hexanes/EtOAc);IR (neat) 3402, 1734, 1662, 1599, 1578, 1510, 1478, 1435, 1354, 1269,1156, 1121, 912, 804, 714 cm⁻¹ ; ¹ H NMR 8.2-8.1 (bs, 1 H), 8.0-7.4 (m,5 H), 5.49 (s, 1 H), 3.86 (s, 3 H), 2.33 (s, 3 H); ¹³ C NMR δ 168.2,167.2, 132.6, 132.5, 132.1, 128.7, 127.3, 83.9, 54.2, 23.2; MS (El) m/e(relative intensity) 235 (13), 208 (18), 192 (8), 121 (7), 105 (100), 77(58).

A solution of 277 mg (1.06 mmol) of triphenylphosphine, 268 mg (1.06mmol) of iodine, and 0.29 mL (2.11 mmol) of triethylamine in 5 mL of CH₂Cl₂ was cooled to -48° C. and treated with a solution of 124 mg (0.528mmol) of 2-benzoylamino-3-oxo-butyric acid methyl ester in 5 mL of CH₂Cl₂. The reaction mixture was warmed to 22° C., stirred for 20 min,transferred to a separatory funnel and extracted with aqueous sodiumthiosuffate followed by saturated sodium bicarbonate. The organic layerwas concentrated in vacuo and chromatographed on SiO₂ (Hexanes/EtOAc,9:1) to give 84.4 mg (74%) of 15 as a white solid: Mp 89.3-89.9° C.(Hexanes/EtOAc); IR (neat) 3025, 1717, 1610, 1561, 1485, 1436, 1348,1323, 1302, 1285, 1235, 1188, 1103, 1072, 1057, 1022 cm⁻¹ ; ¹ H NMR8.1-7.95 (m, 2 H), 7.5-7.3 (m, 3 H), 3.92 (s, 3 H), 2.68 (s, 3 H); ¹³ CNMR δ 162.7, 159.5, 156.3, 130.8, 128.8, 128.6, 128.3, 126.4, 51.9,11.98; MS (El) m/z (relative intensity) 231 (6), 217 (51), 185 (55), 105(100), 77 (4 1), 44 (64); HRMS (El) m/z calculated for C₁₂ H₁₁ NO₃:217.0739, found: 217.0729.

5-Methyl-2-phenyl-oxazole-4-carboxylic acid 11 we produced by stirring asolution of 2.07 g (9.5 mmol) of 15 in 20 mL of 3M NaOH and 12 mL ofMeOH at 22° C. for 2 h and extracting with Et₂ O. The aqueous layer wasacidified to pH 1 with concentrated HCl and extracted with EtOAc. Theorganic layer was dried (Na₂ SO₄), and concentrated in vacuo to give1.84 g (95%) of 11 as an off-white solid: Mp 182.3-182.6° C.(EtOAc/Hexanes); IR (neat) 3200, 2950, 2932, 2890, 2363, 2336, 1694,1682, 1611, 1563, 1483, 1450, 1337, 1255, 1192, 1117, 1053, 1020 cm⁻¹ ;¹ H NMR δ 10.2-9.9 (bs, 1 H), 8.2-7.9 (m, 2 H), 7.6-7.4 (m, 3 H), 2.75(s, 3 H); ¹³ C NMR (CD₃ OD) δ 164.6, 160.7, 157.4, 131.9, 129.8, 129.6,127.3, 127.2, 12.1; MS (El) m/z (relative intensity) 203 (53), 185 (24),157 (13), 116 (17), 105 (100), 89 (21), 77 (33), 63(16); HRMS calculatedfor C₁₁ H₉ NO₃ : 203.0582, found: 203.0583.

2-Decanoylamino-4-(methyl-{3-5-methyl-2-phenyloxazole-4-carbonyl!-ethyl}-carbamoyl)-butyric acidbenzyl ester (formula II) was then provided as follows: To a solution of193 mg (0.363 mmol) of 10 in 15 mL of CH₂ Cl₂ was added 20 mg (0.018mmol) of tetrakistriphenylphosphine Pd(0), 127 μL (0.472 mmol) oftributyltin hydride, and 20 μL of H₂ O. The reaction mixture was stirredat 22° C. for 5 min, filtered through a plug of basic Al₂ O₃ and treatedwith 150 mg (0.726 mmol) of oxazole 11, 60 μL (0.436 mmol) oftriethylamine, and 192 mg (0.436 mmol) of BOP reagent. The reactionmixture was stirred for 30 min at 22° C., diluted with 10 mL of CH₂ Cl₂,and extracted with saturated NaHCO₃ solution, 1M HCl, and brine. Theorganic layer was concentrated in vacuo and chromatographed on SiO₂(Hexanes/EtOAc, 1:1) to give 131 mg (57%) of 2 as a viscous oil: α!_(D)-0.8° (c 1.32, CHCl₃, 21° C.); IR (neat) 3476, 3415, 3311, 3065, 2925,2854, 1741, 1649, 1526, 1491, 1379, 1338, 1264, 1240, 1200, 1174, 1070,711 cm⁻¹ ; ¹ H NMR 8.0-7.95 (m, 2 H), 7.5-7.4 (m, 2 H), 7.33 (bs, 6 H),6.93 (d, 0.3 H, J=7.0 Hz), 6.85 (d, 0.7 H, J=7.2 Hz), 5.18-5.07 (m, 2H), 4.65-4.55 (m, 1 H), 3.7-3.3 (m, 4 H), 2.98 (s, 1 H), 2.96 (s, 2 H),2.71 (d, 3 H, J=2.6 Hz), 2.6-2.0 (m, 6 H), 1.58 (t, 2 H, J=6.8 Hz),1.3-1.1 (bs, 12 H), 0.86 (t, 3 H, J=6.9 Hz) ⁻⁻ C NMR δ 173.3, 172.8,172.0, 171.9, 182.5, 158.6, 153.2, 152.8, 135.9, 130.7, 130.6, 129.7,128.8, 128.5, 128.3, 128.2, 126.7, 126.5, 126.2, 66.9, 52.2, 52.1, 48.9,47.6, 37.2, 37.1, 36.4, 36.3, 36.2, 34.1, 31.8, 29.6, 29.5, 29.4, 29.3,29.2, 28.9, 26.8, 26.6, 25.5, 22.8, 14.1, 11.8; MS (El) m/z (relativeintensity) 632 (38), 497 (9), 405 (18), 374 (22), 260 (21), 220 (42),186 (56), 105 (18), 91 (100); HRMS calculated for C₃₆ H₄₈ N₄ O₆ :632.3574, found: 632.3572.

EXAMPLE II

The solution phase synthesis of compound (formula II) in Example Iestablished the necessary general protocols for the preparation of alibrary of structural variants of the compounds of formula I on a solidsupport. Solid phase synthesis of compounds of formula I is depicted inFIG. 2 and proceeded as follows.

Coupling of diprotected glutamate 5 to the polystyrene-based Wang resindescribed by Wang (1973), J. Am. Chem. Soc. 95:1328, with EDCl wasperformed on large scale and provided a supply of solid phase beads. Thebase-labile Fmoc protective group was removed by treatment withpiperidine and THF, and the resin was distributed to three speciallydesigned Schlenk filters equipped with suction adapters and inert gasinlets for maintaining steady bubbling. After the addition of solvent,hydrophobic residues R'"COCl were added to each flask, which providedthree different amide derivatives 17. After filtration and rinsing ofthe resin, allyl esters 17 were deprotected via Pd(0) chemistry and eachbatch was distributed over three modified Schlenk filters, providingnine different reaction sites for acylation. Addition of three differentN-allyloxycarbonyl protected diamines in the presence of PyBroP³⁹ orCloP⁴⁰ as coupling agents extended the side chain carboxyl terminus ofglutamic acid toward the desired heterocyclic moiety in 1. The resultingnine compounds 18 were each deprotected at the N-terminus anddistributed over two additional Schlenk filters for the final segmentcondensation. Coupling with two different oxazole carboxylic acids inthe presence of CloP and final purification by rinsing with solventprovided the phosphatase library (formula I) still attached to the solidsupport. Complete or partial cleavage with 50% trifluoroacidic acid wasnecessary to release the carboxylate which is required for biologicalactivity. After filtration of the solid support and evaporation of theresulting mother liquor, the desired compounds of formula I wereobtained in a chemically pure and structurally well defined fashionready for rapid throughput biological screening. In each case, thepurity of the final compound was >60% according to spectroscopicanalysis (¹ H NMR, MS). The contamination was derived from incompletecouplings to the sterically hindered secondary amine moiety ofAlloc-NHCH₂ CH₂ NH(R").

The foregoing synthesis is provided in more detail as follows:

Step 1, 5→16. In a medium porosity Schlenk filter apparatus was placed750 mg of Wang resin (0.96 mmol/g, 0.72 mmol of active sites). The resinwas suspended in 12 mL of dry DMF and a stream of nitrogen was forced upthrough the filter at a rate which allowed the solvent to gently bubble.To this reaction mixture was added 1.47 g (3.6 mmol) of 5. Thesuspension was agitated for 5 min and treated with 26 mg (0.216 mmol) ofDMAP and 550 mg (2.88 mmol) of EDCl, agitated at 22° C. for 18 h andfiltered, and the resin was washed with DMF (2×10 mL), H₂ O (3×10 mL),THF (3×10 mL), and CH₂ Cl₂ (3×10 mL). The resin was dried under vacuumand the remaining active sites were capped by addition of 10 mL of CH₂Cl₂ and 10 mL of acetic anhydride along with 26 mg (2.88 mmol) of DMAPto the resin. Bubbling was continued at 22° C. for 3 h and the resin wasthen washed with CH₂ Cl₂ (6×15 mL) and dried in vacuo. To test theloading on the resin, 30 mg of resin was removed and suspended in 2 mLof trifluoroacetic acid for 5 min at 22° C., filtered and washed (3×3mL) with CH₂ Cl₂. The filtrate was concentrated in vacuo to give 7.3 mg(85%) of 5.

Step 2, 16→17. A suspension of 690 mg (0.576 mmol) of2-(9H-fluoren-9-ylmethoxycarbonylamino) pentanedioic acid 5-allyl esterlinked to Wang resin 16 in 15 mL of THF was treated with 6 mL (57.6mmol) of piperidine, agitated by bubbling for 30 min, filtered andwashed with CH₂ Cl₂ (6×10 mL). The resin was dried in vacuo. Asuspension of this resin in 10 mL of CH₂ Cl₂ was treated with 0.48 mL(2.31 mmol) of decanoyl chloride and 14 mg (0.115 mmol) of DMAP. Thereaction mixture was agitated at 22° C. for 6 h, filtered and the resinwas washed with CH₂ C₂ (6×10 mL) and dried in vacuo.

Step 3, 17→18. A suspension of 690 mg (0.576 mmol) of2-decanoylamino-pentanedioic acid 5-allyl ester linked to Wang resin 17in 10 mL of THF was treated with 67 mg (0.0576 mmol) oftetrakis(triphenylphosphine) palladium (0) and 806 mg (5.75 mmol) ofdimedone, and agitated by bubbling at 22° C. for 18 h. The resin wasthen filtered, washed with THF (2×10 mL), CH₂ Cl₂ (2×10 mL), MeOH (2×10mL), H₂ O (2×10 mL), 1% acetic acid solution (2×10 mL), H₂ O (2×10 mL),MeOH (2×10 mL), CH₂ Cl₂ (2×10 mL), and dried in vacuo. Cleavage andexamination of 40 mg of resin by ¹ H NMR showed full deprotection of theallyl ester.

A suspension of this resin in 12 mL of DMF was treated with 0.22 mL(1.572 mmol) of triethylamine and 414.1 mg (2.62 mmol) of Alloc-NHCH₂CH₂ NHMe. After agitating the reaction mixture for 5 min to ensureproper mixing, 540 mg (1.572 mmol) of CloP was added. The reactionmixture was agitated with bubbling for 18 h at 30° C., cooled to 22° C.,and the resin was filtered and washed with DMF (2×10 mL), CH₂ Cl₂ (2×10mL), MeOH (2×10 mL), H₂ O (2×10 mL), THF (2×10 mL), and CH₂ Cl₂ (2×10mL). The resin was dried in vacuo and 40 mg of resin was cleaved withCF₃ CO₂ H. The ¹ H NMR of the residue showed that coupling had occurredto nearly 100%.

Step 5, 18→19. A suspension of 200 mg (0.192 mmol) of 4-(2-allyloxycarbonylaminoethyl)-methyl-carbamoyl!-2-decanoylamino-butyricacid linked to Wang resin 18 in 6 mL of CH₂ Cl₂ was treated with 12 mg(0.0096 mmol) of tetrakistriphenylphosphine Pd(0), 62 μl (0.230 mmol) oftributyltin hydride, and 10 μl of H₂ O. The reaction mixture wasagitated with bubbling N₂ for 15 min, filtered, and the resin was washedwith 10 mL portions of CH₂ Cl₂, THF, acetone, MeOH, H₂ O, acetone,EtOAc, hexanes, THF, and CH₂ Cl₂. The resin was then dried in vacuo and15 mg was removed for testing. The ¹ H NMR of the TFA-cleaved residueshowed full deprotection as well as full removal of all tin sideproducts.

A suspension of 185 mg (0.190 mmol) of this resin in 8 mL of CH₂ Cl₂ wastreated with 117 mg (0.576 mmol) of oxazole carboxylic acid, 198 mg(0.576 mg) of CloP, and 80 μl (0.576 mmol) of triethylamine. Thereaction mixture was agitated by bubbling with N₂ for 3 h, filtered, andwashed with 20 mL of CH₂ Cl₂, acetone, water, acetone, and CH₂ Cl₂. Theresin was dried in vacuo and 15 mg was removed for testing. The ¹ H NMRof the residue showed that the reaction had gone to 60% completion. Theresin was subsequently submitted to a second coupling cycle.

Step 6, 19→1. A suspension of 115 mg (0.12 mmol) of2-decanoylamino-4-(methyl-{3-5-methyl-2-phenyl-oxazole-4-carbonyl!-ethyl}-carbamoyi)-butyric acidlinked to Wang resin 19 in 3 mL of TFA was stirred for 5 min, filtered,and washed with 5 mL of CH₂ Cl₂. The extract was concentrated in vacuoto provide 33.1 mg (100% for step 2 to step 6) of 1. A ¹ H NMR showedthe product to be 66% pure with 2-acylamino-pentanedioic acid as themajor impurity. Acid 1a was dissolved in 3 mL of CH₂ Cl₂ and treatedwith 0.016 mL (0.138 mmol) of benzyl bromide and 0.02 mL (0.138 mmol) ofDBU to provide material identical with the benzyl ester 2 prepared bysolution phase chemistry.

EXAMPLE III

The following compounds 1a-r (Table 1) corresponding to formula I weretested for their ability to inhibit PP1, PP2A and PP3.

                  TABLE 1    ______________________________________    Compound   R      R'        R"    R'"    ______________________________________    1a         Ph     CH.sub.3  CH.sub.3                                      n-C.sub.9 H.sub.19    1b         Ph     CH.sub.3  n-C.sub.6 H.sub.13                                      n-C.sub.9 H.sub.19    1c         Ph     CH.sub.3  Bn    n-C.sub.9 H.sub.19    1d         Ph     Ph        CH.sub.3                                      n-C.sub.9 H.sub.19    1e         Ph     Ph        n-C.sub.6 H.sub.13                                      n-C.sub.9 H.sub.19    1f         Ph     Ph        Bn    n-C.sub.9 H.sub.19    1g         Ph     CH.sub.3  CH.sub.3                                      PhCH.sub.2 CH.sub.2    1h         Ph     CH.sub.3  n-C.sub.6 H.sub.13                                      PhCH.sub.2 CH.sub.2    1i         Ph     CH.sub.3  Bn    PhCH.sub.2 CH.sub.2    1j         Ph     Ph        CH.sub.3                                      PhCH.sub.2 CH.sub.2    1k         Ph     Ph        n-C.sub.6 H.sub.13                                      PhCH.sub.2 CH.sub.2    11         Ph     Ph        Bn    PhCH.sub.2 CH.sub.2    1m         Ph     CH.sub.3  CH.sub.3                                      PhCH=CH    1n         Ph     CH.sub.3  n-C.sub.6 H.sub.13                                      PhCH=CH    1o         Ph     CH.sub.3  Bn    PhCH=CH    1p         Ph     Ph        CH.sub.3                                      PhCH=CH    1q         Ph     Ph        n-C.sub.6 H.sub.13                                      PhCH=CH    1r         Ph     Ph        Bn    PhCH=CH    ______________________________________

Phosphatase activity and the inhibitory activity of the compounds ofTable 1 were determined by the method of Honkanen et al. (1994) Toxicon32:339. Briefly, phosphatase activity against phosphorylase-a orphosphohistone was determined by the quantification of liberated ³² P!.Assays, 80 μl total volume, containing 50 mM Tris-HCl, pH 7.4, 0.5 mMDTT, 1 mM EDTA (assay buffer) and ³² P! phosphoprotein (1-2 μM PO₄),were conducted as described previously (Honkanen et al., 1991 Mol.Pharmac. 40:577). Dephosphorylation reactions were routinely conductedfor 5-10 min. using phosphorylase-a as a substrate and for 10-20 min.using phosphohistone. In all assays the dephosphorylation of substratewas kept to less than 10% of the total phosphorylated substrateavailable, and the reactions were adjusted to ensure that enzymeactivity was linear with respect to enzyme concentration and time. ³² P!Phosphate liberated by the enzymes was extracted as a phosphomolybdatecomplex and measured according to the methods of Killilea et al. (1978)Arch. Biochem. Biophys. 191.638. Inhibition of protein phosphataseactivity by inhibitors was determined by adding the 100 μM of inhibitorsto the enzyme mixture 5-10 min. prior to initiating the reaction withthe addition of substrate.

Results are presented in Table 2 as the percent inhibition relative tocontrol (100%).

                  TABLE 2    ______________________________________    Compound   PP1           PP2A   PP3    ______________________________________    none       100           100    100    1a         121           100    76    1b         59            135    32    1c         ND            129    28    1d         53            69     33    1e         153           152    71    1f         117           156    85    1g         53            109    21    1h         63            88     17    1i         ND            80     14    1j         80            72     38    1k         48            67     24    1L         59            69     22    1m         111           108    60    1n         40            87     26    1o         64            88     13    1p         65            99     68    1q         85            60     28    1r         53            68     15    ______________________________________

Compound 1d (R=Ph, R'=Ph, R"=CH₃, R'"=n-C₉ H₁₉) was further assessed forits ability to inhibit PP2A, and compared to calyculin A, a knowninhibitor of PP2A.

The activity of the catalytic subunit of bovine cardiac muscle PP2A(Gibco-BRL, Gaithersburg, Md.) was measured with fluorescein diphosphate(Molecular Probes, Inc., Eugene, OR) as a substrate in 96-wellmicrotiter plates. The final incubation mixture (150 μL) composed 25 mMTris (pH=7.5), 5 mM EDTA, 33 μg/mL BSA, and 20 μM fluoresceindiphosphate. Inhibitors were resuspended in DMSO, which was also used asthe vehicle control. Reactions were initiated by adding 0.2 units ofPP2A and incubated at room temperature overnight. Fluorescence emissionfrom the product was measured with Perseptive Biosystems Cytoflour II(exciton filter, 485 nm; emission filter, 530 nm) (Framingham, Mass.).

As demonstrated in FIG. 6, calyculin A inhibited PP2A activity at 10 nM,and compound id caused 50% inhibition at 100 μM.

EXAMPLE IV

Compounds 1a-1r were assessed for ability to inhibit CDC25A and CDC25Bactivity. Recombinant human CDC25A and CDC25B were obtained as aglutahione-S-transferase (GST) fusion protein using human cDNA andstandard molecular biological methods. The cDNA constructs are in aplasmid that is expressed in E. coli under the control ofisopropyl-beta-D-thiogalactosidase (IPTG). The bacterial pellet wasdisrupted by sonication, and centrifuged at 10,000×g. Usingglutathione-agarose beads, the fusion protein was purified frompostmicrosomal supernant fraction as described by Baratte et al.(1992)Anticancer Res. 12: 873-880. Phosphatase activity was assayed with aspectrofluorimeter under the following conditions: 1 unit (1 U=amount ofprotein that induces 33 fluoresence units/minute of product) of fusionprotein in a final incubation mixture (150 μL) comprised of 25 mM Tris(pH=8.0), 5 mM EDTA, 33 μg/ml BSA, and 20 μM fluorescein diphosphate in96-well microtiter plates. Plates were preincubated for 1 hour with 0(control), 0.3, 1, 3, 10, 30, 100 μM compounds at room temperature.After the 1 hour incubation at room temperature, fluorescence of thefluorescein product (Ex. 485 nm; Em. 530 nm) were measured with aBiosystems Cytofluor II (Framingham, Mass.).

Results are presented in FIG. 7 and Table 3 as percent inhibitionrelative to control. A dose-response curve for compound 1f is presentedin FIG. 8.

                  TABLE 3    ______________________________________    Compound        CDC25A   CDC25B    ______________________________________    none            100      100    1a              67       106    1b              14       44    1c              11       42    1d              99       91    1e               7       17    1f               3       15    1g              50       81    1h              36       69    1i              31       63    1j              107      90    1k              83       76    1L              62       69    1m              19       45    1n              29       66    1o              35       66    1p              71       87    1q              55       62    1r              42       56    ______________________________________

The foregoing results demonstrate that compounds 1a-1c and 1e-1r arecapable of inhibiting the activity of CDC25A and/or CDC25B.

EXAMPLE V

Compounds 1a-1r were tested for antiproliferative activity against humanMDA-MB-231 breast cancer cells.

Human MDA-MB-231 breast carcinoma cells were obtained from the AmericanType Culture Collection at passage 28 and were maintained for no longerthan 20 passages. The cells were grown in RPMI-1640 supplemented with 1%penicillin (100 μg/mL) and streptomycin (100 μg/mL), 1% L-glutamate, and10% fetal bovine serum in a humidified incubator at 37° C. under 5% CO₂in air. Cells were routinely found free of mycoplasma. To remove cellsfrom the monolayer for passage or flow cytometry, cells were washed twotimes with phosphate buffer and briefly (<3 min) treated with 0.05%trypsin/2 mM EDTA at room temperature. After the addition of at leasttwo volumes of growth medium containing 10% fetal bovine serum, thecells were centrifuged at 1,000×g for 5 min. Compounds were made intostock solutions using DMSO, and stored at -20° C. All compounds andcontrols were added to obtain a final concentration of 0.1-0.2% (v/v) ofthe final solution for experiments.

The antiproliferative activity of the compounds was determined by themethod of Lazo et al. (1995) J. Biol. Chem. 270:5506. Briefly, cells(6.5×10³ cells/cm²) were plated in 96 well flat bottom plates for thecytotoxicity studies and incubated at 37° C. for 48 h. The platingmedium was aspirated off 96 well plates and 200 μL of growth mediumcontaining the compound was added per well. Compounds were used at from0 to the highest available concentration which ranged from 30 to 100 μM.Plates were incubated for 72 h, and then washed 4× with serum freemedium. After washing, 50 μL of 3-4,5-dimethylthiazol-2-yl!-2,5-diphenyl tetrazolium bromide solution (2mg/mL) was added to each well, followed by 150 μL of complete growthmedium. Plates were then incubated an additional 4 h at 37° C. Thesolution was aspirated off, 200 μL of DMSO added, and the plates wereshaken for 30 min at room temperature. Absorbance at 540 nm wasdetermined with a Titertek Multiskan Plus plate reader. Biologicallyactive compounds were tested at least 3 independent times.

Administration of compound 1h caused 50% growth inhibition at 20 μM buthad no further cytotoxicity at higher drug concentrations. Compound ifcaused 50% growth inhibition at 100 μM and had a clearconcentration-dependency (FIG. 9).

EXAMPLE VI

The cell cycle distribution of human breast cancer cells after treatmentwith compound if was determined by flow cytometry.

MDA-MB-231 cells (6.5×10⁵ /cm²) were plated and incubated at 37° C. for48 h. The plating medium was then aspirated off, and medium containing aconcentration of compound if that caused approximately 50% growthinhibition (88-100 μM) was added for 48 to 72 h. Untreated cells at asimilar cell density were used as control populations. Single cellpreparations were fixed in ice-cold 1% paraformaldehyde, centrifuged at1,000×g for 5 min, resuspended in Puck's saline, centrifuged, andresuspended in ice-cold 70% ethanol overnight. The cells were removedfrom fixatives by centrifugation (1,000×g for 5 min) and stained with a5 μg/mL propidium iodide and 50 μg/mL RNase A solution. Flow cytometryanalyses were conducted with a Becton Dickinson FACS Star. Singleparameter DNA histograms were collected for 10,000 cells, and cell cyclekinetic parameters calculated using DNA cell cycle analysis softwareversion C (Becton Dickinson). Experiments at 72 h were performed atleast 3 independent times.

Results are presented in FIGS. 10A-D.

Exponentially growing human MDA-MB-231 breast cancer cell populations(population doubling time of approximately 30-35 h) typically haveapproximately 30% of all cells in the S or DNA synthetic phase of thecell cycle (FIGS. 10A and C). In contrast, when MDA-MB-231 cells wereincubated for 48 h with 88 μM compound if, there was prominentaccumulation in the G1 phase with a concomitant decrease in both S andG2/M phases (FIGS. 10B and C). Incubation of MDA-MB-231 cells for 72 hwith 88 μM if also caused a prominent accumulation in the G1 phase (FIG.10D).

These results demonstrate that compound 1f exhibits aconcentration-dependent inhibition in proliferation of MDA-MB-231 cells,and that blockage in cell cycle progression is at the G1 checkpoint.

We claim:
 1. A method of inhibiting a protein phosphatase comprisingcontacting the protein phosphatase with a protein phosphatase-inhibitingeffective amount of a compound having the formula ##STR9## wherein R,R', R", and R'" are independently H, alkyl, alkenyl, alkynyl,cycloalkyl, phenyl, oxetanyl, azetidinyl, furanyl, pyrrole, indolyl,oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl,pyranyl, pyridyl, pyridonyl, piperidyl, piperazinyl, quinolyl, azepinyl,and diazepinyl.
 2. The method of claim 1 wherein said proteinphosphatase is a serine/threonine protein phosphatase.
 3. The method ofclaim 2 wherein said protein phosphatase is PP1, PP2A, or PP3.
 4. Themethod of claim 1 wherein said protein phosphatase is a dual specificityphosphatase.
 5. The method of claim 4 wherein said phosphatase is CDC25Aor CDC25B.